Rb and N-Ras Function Together to Control Differentiation in the Mouse

Total Page:16

File Type:pdf, Size:1020Kb

Rb and N-Ras Function Together to Control Differentiation in the Mouse Rb and N-ras Function Together to Control Differentiation in the Mouse The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Takahashi, C., R. T. Bronson, M. Socolovsky, B. Contreras, K. Y. Lee, T. Jacks, M. Noda, R. Kucherlapati, and M. E. Ewen. 2003. “Rb and N-Ras Function Together To Control Differentiation in the Mouse.” Molecular and Cellular Biology 23 (15): 5256–68. doi:10.1128/ MCB.23.15.5256-5268.2003. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:41543053 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA MOLECULAR AND CELLULAR BIOLOGY, Aug. 2003, p. 5256–5268 Vol. 23, No. 15 0270-7306/03/$08.00ϩ0 DOI: 10.1128/MCB.23.15.5256–5268.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved. Rb and N-ras Function Together To Control Differentiation in the Mouse Chiaki Takahashi,1† Roderick T. Bronson,2,3 Merav Socolovsky,4 Bernardo Contreras,1 Kwang Youl Lee,1‡ Tyler Jacks,5 Makoto Noda,6 Raju Kucherlapati,7 and Mark E. Ewen1* Department of Medical Oncology and Medicine, Dana-Farber Cancer Institute and Harvard Medical School,1 and Rodent Histopathology Core3 and Harvard-Partners Center for Genetics and Genomics,7 Harvard Medical School, Boston, Massachusetts 02115; Department of Pathology, Tufts University Schools of Medicine and Veterinary Medicine, 2 Boston, Massachusetts 02111 ; Whitehead Institute for Biomedical Research, Massachusetts Institute of Downloaded from Technology, Cambridge, Massachusetts 021424; Department of Biology and Howard Hughes Medical Institute, Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 021395; and Department of Molecular Oncology, Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto 606-8501, Japan6 Received 8 November 2002/Returned for modification 7 January 2003/Accepted 30 April 2003 The product of the retinoblastoma tumor suppressor gene (Rb) can control cell proliferation and promote dif- ferentiation. Murine embryos nullizygous for Rb die midgestation with defects in cell cycle regulation, control http://mcb.asm.org/ of apoptosis, and terminal differentiation of several tissues, including skeletal muscle, nervous system, and lens. Previous cell culture-based experiments have suggested that the retinoblastoma protein (pRb) and Ras operate in a common pathway to control cellular differentiation. Here we have tested the hypothesis that the proto-oncogene N-ras participates in Rb-dependent regulation of differentiation by generating and characterizing murine em- bryos deficient in both N-ras and Rb. We show that deletion of N-ras rescues a unique subset of the develop- mental defects associated with nullizygosity of Rb, resulting in a significant extension of life span. Rb؊/؊;N-ras؊/؊ skeletal muscle has normal fiber density, myotube length and thickness, in contrast to Rb-deficient embryos. Additionally, Rb؊/؊;N-ras؊/؊ muscle shows a restoration in the expression of the late muscle-specific gene ,MCK, and this correlates with a significant potentiation of MyoD transcriptional activity in Rb؊/؊;N-ras؊/؊ ؊/؊ ؊/؊ ؊/؊ compared to Rb myoblasts in culture. The improved differentiation of skeletal muscle in Rb ;N-ras on October 13, 2019 by guest embryos occurs despite evidence of deregulated proliferation and apoptosis, as seen in Rb-deficient animals. Our findings suggest that the control of differentiation and proliferation by Rb are genetically separable. The loss of tumor suppressor gene function is a common through its interaction with E2F, and its association with other event in the development of human cancer. The retinoblas- factors involved in altering chromatin structure (e.g., histone toma gene (Rb) has served as the paradigm for the study of this deacetylase and BRG1) leads to active repression of genes class of genes (67, 68). Mutations resulting in the inactivation involved in cell proliferation (e.g., cyclin E). The ability of pRb to of Rb are found in a large fraction of human cancers of both influence cell death also appears to involve its regulation of mesenchymal and epithelial origin (57, 62). An understanding E2F (13, 56, 63). of how the retinoblastoma protein (pRb) exerts its tumor- How pRb regulates differentiation is poorly understood. suppressive action can be gained from knowledge of the bio- Most differentiation programs involve withdrawal from the cell logical and molecular consequences of its inactivation. cycle, but the participation of pRb appears to extend beyond pRb participates in the control of cell cycle progression, an ability to merely facilitate the process by inhibiting E2F and apoptosis, and differentiation. How it exerts effects on prolif- cell cycle progression. Naturally occurring mutants of pRb can eration is well understood. This appears to be achieved by its be identified that retain tumor suppressor activity and the regulated interaction with the E2F family of transcription fac- ability to promote differentiation when assayed in vitro despite tors (14, 56, 63). E2F can bind to and promote the expression having lost the capability to bind to E2F (28, 58). Consistent of a number of genes involved in cell cycle progression (e.g., with this, pRb influences the activity of a number of transcrip- DHFR). pRb, by binding to E2F, can inhibit the transactivation tion factors known to participate in different differentiation function of E2F. In addition, pRb is targeted to promoters processes, such as MyoD, the glucocorticoid receptor, CBFA1, and C/EBP␤ (3, 4, 11, 43, 44, 59, 61). Also, cell culture studies * Corresponding author. Mailing address: Department of Medical have demonstrated a key role for pRb in myogenesis, osteo- Oncology and Medicine, Dana-Farber Cancer Institute and Harvard genic differentiation, and adipogenesis (3, 6, 11, 43, 54, 58, 61). Medical School, Boston, MA 02115. Phone: (617) 632-2206. Fax: (617) Mouse genetics have been employed to better understand 632-5417. E-mail: [email protected]. the physiological functions of pRb (35, 66). Mice heterozygous † Present addresses: Department of Molecular Oncology, Kyoto Uni- for Rb succumb to pituitary tumors (15, 17, 19), while inacti- versity Graduate School of Medicine, Sakyo-ku, Kyoto 606-8501, Japan. ‡ Present address: Chung-buk National University, College of Med- vation of both Rb alleles results in embryos that die in mid- icine, Cheong-Ju, Korea. gestation (5, 19, 31, 71). These embryos are characterized by 5256 VOL. 23, 2003 GENETIC INTERACTION BETWEEN Rb AND N-ras 5257 defects in erythroid, neuronal, and skeletal muscle differenti- and cell death. Our analyses suggest that N-ras operates with ation, and ectopic S-phase entry and apoptosis are observed in Rb in the control of cellular differentiation. the central nervous system (CNS), peripheral nervous system (PNS), lens, and skeletal muscle (5, 19, 31, 32, 71). The con- MATERIALS AND METHODS tribution of deregulated E2F activity to these phenotypes has Mouse strains. Parental Rbϩ/Ϫ and N-rasϩ/Ϫ mice were maintained on a been assessed with compound embryos lacking Rb and E2f-1 or mixed genetic background (C57BL/6 ϫ 129/Sv and C57BL/6 ϫ 129/Ola, respec- ϩ/Ϫ ϩ/Ϫ E2f-3 (21, 64, 72). These embryos live longer than their RbϪ/Ϫ tively) and intercrossed to generate subsequent founders. Rb ;N-ras fe- males were crossed with Rbϩ/Ϫ;N-rasϩ/ϩ, Rbϩ/Ϫ;N-rasϩ/Ϫ,orRbϩ/Ϫ;N-rasϪ/Ϫ counterparts, and this has been attributed to a partial restora- males. Timed pregnancies were established by the detection of a plug, taken as tion of fetal liver erythropoiesis. Additionally, loss of either embryonic day 0.5 (E0.5). Mice and embryos were genotyped by PCR with E2f-1 or E2f-3 can suppress the deregulated proliferation and genomic DNA extracted from tails and yolk sacs, respectively, as previously apoptosis to significant but varying degrees in the CNS, PNS, described (19, 65). All animal experimentation was performed at the Dana- Farber Cancer Institute Animal Resource Facility in accordance with the guide- and lens. Importantly, the lens of Rb-deficient embryos also lines of the National Institutes of Health. shows signs of aberrant differentiation that are not rescued by Histology and immunohistochemistry. Embryos were fixed in Bouin’s solution, Downloaded from loss of E2f-1, suggesting again that Rb’s influence on cell cycle rinsed with 70% ethanol, and embedded in paraffin for sectioning. Sections (6 progression and apoptosis is genetically separable from its ␮m) were stained with hematoxylin and eosin (H&E). Alternatively, sections regulation of differentiation (36). A notable aspect of these were incubated with a monoclonal antibody (MY-32; Sigma) to myosin heavy chain (MHC) following deparaffinization and rehydration. To identify prolifer- Ϫ/Ϫ studies is the observation that the extended life span of Rb ; ating cells, bromodeoxyuridine (BrdU) was injected intraperitoneally (33 ␮g per Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ E2f-1 / and Rb / ; E2f-3 / embryos reveals additional phe- mouse) 1 h prior to sacrifice. Fixed embryos were rinsed with 70% ethanol and notypes, including developmental defects in the lung and embedded in paraffin, from which sections were cut. After rehydration, the heart, and more pronounced defects in skeletal muscle. Ab- endogenous peroxidase activity was quenched with 3% H2O2–10% methanol in phosphate-buffered saline (PBS) (pH 7.4). Sections were then treated succes- normalities in skeletal muscle differentiation are also observed sively with 0.05 mM trypsin, 2 N HCl, and PBS (pH 6.0). After blocking with 6% in Rb-deficient mice with a partial reconstitution of Rb (71) goat serum, sections were incubated with an anti-BrdU mouse monoclonal an- http://mcb.asm.org/ and in RbϪ/Ϫ; Id2Ϫ/Ϫ mice (30). Indeed, respiratory failure tibody (B44; Becton Dickinson) in the presence of 0.5% Tween 20 in PBS (pH resulting from the complete lack of muscle fibers in the dia- 7.4).
Recommended publications
  • Retinoblastoma Protein Positively Regulates Terminal Adipocyte Differentiation Through Direct Interaction with C/Ebps
    Downloaded from genesdev.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press Retinoblastoma protein positively regulates terminal adipocyte differentiation through direct interaction with C/EBPs Phang-Lang Chen, Daniel J. Riley, Yumay Chen, and Wen-Hwa Lee l Center for Molecular Medicine, Institute of Biotechnology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78245 USA To define a mechanism by which retinoblastoma protein (Rb) functions in cellular differentiation, we studied primary fibroblasts from the lung buds of wild-type (RB + / +) and null-mutant (RB-/-) mouse embryos. In culture, the RB +/+ fibroblasts differentiated into fat-storing cells, either spontaneously or in response to hormonal induction; otherwise syngenic RB -/- fibroblasts cultured in identical conditions did not. Ectopic expression of normal Rb, but not Rb with a single point mutation, enabled RB-/- fibroblasts to differentiate into adipocytes. Rb appears in murine fibroblasts to activate CCAAT/enhancer-binding proteins (C/EBPs), a family of transcription factors crucial for adipocyte differentiation. Physical interaction between Rb and C/EBPs was demonstrated by reciprocal coimmunoprecipitation, but occurred only in differentiating cells. Wild-type Rb also enhanced the binding of C/EBP to cognate DNA sequences in vitro and the transact[vat[on of a C/EBPl$-responsive promoter in cells. Taken together, these observations establish a direct and positive role for Rb in terminal differentiation. Such a role contrasts with the function of Rb in arresting cell cycle progression in G1 by negative regulation of other transcription factors like E2F-1. [Key Words: C/EBP; transcription factors; cell cycle; adipocyte differentiation; tumor suppressor protein] Received July 18, 1996; revised version accepted September 5, 1996.
    [Show full text]
  • Prox1regulates the Subtype-Specific Development of Caudal Ganglionic
    The Journal of Neuroscience, September 16, 2015 • 35(37):12869–12889 • 12869 Development/Plasticity/Repair Prox1 Regulates the Subtype-Specific Development of Caudal Ganglionic Eminence-Derived GABAergic Cortical Interneurons X Goichi Miyoshi,1 Allison Young,1 Timothy Petros,1 Theofanis Karayannis,1 Melissa McKenzie Chang,1 Alfonso Lavado,2 Tomohiko Iwano,3 Miho Nakajima,4 Hiroki Taniguchi,5 Z. Josh Huang,5 XNathaniel Heintz,4 Guillermo Oliver,2 Fumio Matsuzaki,3 Robert P. Machold,1 and Gord Fishell1 1Department of Neuroscience and Physiology, NYU Neuroscience Institute, Smilow Research Center, New York University School of Medicine, New York, New York 10016, 2Department of Genetics & Tumor Cell Biology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105, 3Laboratory for Cell Asymmetry, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan, 4Laboratory of Molecular Biology, Howard Hughes Medical Institute, GENSAT Project, The Rockefeller University, New York, New York 10065, and 5Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724 Neurogliaform (RELNϩ) and bipolar (VIPϩ) GABAergic interneurons of the mammalian cerebral cortex provide critical inhibition locally within the superficial layers. While these subtypes are known to originate from the embryonic caudal ganglionic eminence (CGE), the specific genetic programs that direct their positioning, maturation, and integration into the cortical network have not been eluci- dated. Here, we report that in mice expression of the transcription factor Prox1 is selectively maintained in postmitotic CGE-derived cortical interneuron precursors and that loss of Prox1 impairs the integration of these cells into superficial layers. Moreover, Prox1 differentially regulates the postnatal maturation of each specific subtype originating from the CGE (RELN, Calb2/VIP, and VIP).
    [Show full text]
  • And Represses TNF Gene Expression Activating Transcription Factor-2
    Histone Deacetylase 3, a Class I Histone Deacetylase, Suppresses MAPK11-Mediated Activating Transcription Factor-2 Activation and Represses TNF Gene Expression This information is current as of September 25, 2021. Ulrich Mahlknecht, Jutta Will, Audrey Varin, Dieter Hoelzer and Georges Herbein J Immunol 2004; 173:3979-3990; ; doi: 10.4049/jimmunol.173.6.3979 http://www.jimmunol.org/content/173/6/3979 Downloaded from References This article cites 45 articles, 31 of which you can access for free at: http://www.jimmunol.org/content/173/6/3979.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 25, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Histone Deacetylase 3, a Class I Histone Deacetylase, Suppresses MAPK11-Mediated Activating Transcription Factor-2 Activation and Represses TNF Gene Expression1 Ulrich Mahlknecht,2* Jutta Will,* Audrey Varin,† Dieter Hoelzer,* and Georges Herbein2† During inflammatory events, the induction of immediate-early genes, such as TNF-␣, is regulated by signaling cascades including the JAK/STAT, NF-␬B, and the p38 MAPK pathways, which result in phosphorylation-dependent activation of transcription factors.
    [Show full text]
  • Targeting Endothelial Kruppel-Like Factor 2 (KLF2) in Arteriovenous
    Targeting Endothelial Krüppel-like Factor 2 (KLF2) in Arteriovenous Fistula Maturation Failure A dissertation submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY (Ph.D.) in the Biomedical Engineering Program Department of Biomedical Engineering College of Engineering and Applied Science 2018 by Keith Louis Saum B.S., Wright State University, 2012 Dissertation Committee: Albert Phillip Owens III, Ph.D. (Committee Chair) Begona Campos-Naciff, Ph.D Christy Holland, Ph.D. Daria Narmoneva, Ph.D. Prabir Roy-Chaudhury, M.D., Ph.D Charuhas Thakar, M.D. Abstract The arteriovenous fistula (AVF) is the preferred form of vascular access for hemodialysis. However, 25-60% of AVFs fail to mature to a state suitable for clinical use, resulting in significant morbidity, mortality, and cost for end-stage renal disease (ESRD) patients. AVF maturation failure is recognized to result from changes in local hemodynamics following fistula creation which lead to venous stenosis and thrombosis. In particular, abnormal wall shear stress (WSS) is thought to be a key stimulus which alters endothelial function and promotes AVF failure. In recent years, the transcription factor Krüppel-like factor-2 (KLF2) has emerged as a key regulator of endothelial function, and reduced KLF2 expression has been shown to correlate with disturbed WSS and AVF failure. Given KLF2’s importance in regulating endothelial function, the objective of this dissertation was to investigate how KLF2 expression is regulated by the hemodynamic and uremic stimuli within AVFs and determine if loss of endothelial KLF2 is responsible for impaired endothelial function.
    [Show full text]
  • Nuclear Localization of DP and E2F Transcription Factors by Heterodimeric Partners and Retinoblastoma Protein Family Members
    Journal of Cell Science 109, 1717-1726 (1996) 1717 Printed in Great Britain © The Company of Biologists Limited 1996 JCS7086 Nuclear localization of DP and E2F transcription factors by heterodimeric partners and retinoblastoma protein family members Junji Magae1, Chin-Lee Wu2, Sharon Illenye1, Ed Harlow2 and Nicholas H. Heintz1,* 1Department of Pathology, University of Vermont, Burlington VT 05405, USA 2Massachusetts General Hospital Cancer Center, Charlestown MA 02129, USA *Author for correspondence SUMMARY E2F is a family of transcription factors implicated in the showed that regions of E2F-1 and DP-1 that are required regulation of genes required for progression through G1 for stable association of the two proteins were also required and entry into the S phase. The transcriptionally active for nuclear localization of DP-1. Unlike E2F-1, -2, and -3, forms of E2F are heterodimers composed of one polypep- E2F-4 did not accumulate in the nucleus unless it was coex- tide encoded by the E2F gene family and one polypeptide pressed with DP-2. p107 and p130, but not pRb, stimulated encoded by the DP gene family. The transcriptional activity nuclear localization of E2F-4, either alone or in combina- of E2F/DP heterodimers is influenced by association with tion with DP-2. These results indicate that DP proteins the members of the retinoblastoma tumor suppressor preferentially associate with specific E2F partners, and protein family (pRb, p107, and p130). Here the intracellu- suggest that the ability of specific E2F/DP heterodimers to lar distribution of E2F and DP proteins was investigated in localize in the nucleus contributes to the regulation of E2F transiently transfected Chinese hamster and human cells.
    [Show full text]
  • Global Mef2 Target Gene Analysis in Skeletal and Cardiac Muscle
    GLOBAL MEF2 TARGET GENE ANALYSIS IN SKELETAL AND CARDIAC MUSCLE STEPHANIE ELIZABETH WALES A DISSERTATION SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY GRADUATE PROGRAM IN BIOLOGY YORK UNIVERSITY TORONTO, ONTARIO FEBRUARY 2016 © Stephanie Wales 2016 ABSTRACT A loss of muscle mass or function occurs in many genetic and acquired pathologies such as heart disease, sarcopenia and cachexia which are predominantly found among the rapidly increasing elderly population. Developing effective treatments relies on understanding the genetic networks that control these disease pathways. Transcription factors occupy an essential position as regulators of gene expression. Myocyte enhancer factor 2 (MEF2) is an important transcription factor in striated muscle development in the embryo, skeletal muscle maintenance in the adult and cardiomyocyte survival and hypertrophy in the progression to heart failure. We sought to identify common MEF2 target genes in these two types of striated muscles using chromatin immunoprecipitation and next generation sequencing (ChIP-seq) and transcriptome profiling (RNA-seq). Using a cell culture model of skeletal muscle (C2C12) and primary cardiomyocytes we found 294 common MEF2A binding sites within both cell types. Individually MEF2A was recruited to approximately 2700 and 1600 DNA sequences in skeletal and cardiac muscle, respectively. Two genes were chosen for further study: DUSP6 and Hspb7. DUSP6, an ERK1/2 specific phosphatase, was negatively regulated by MEF2 in a p38MAPK dependent manner in striated muscle. Furthermore siRNA mediated gene silencing showed that MEF2D in particular was responsible for repressing DUSP6 during C2C12 myoblast differentiation. Using a p38 pharmacological inhibitor (SB 203580) we observed that MEF2D must be phosphorylated by p38 to repress DUSP6.
    [Show full text]
  • RB1 and P53 at the Crossroad of EMT and Triple Negative Breast Cancer
    PERSPECTIVE PERSPECTIVE Cell Cycle 10:10, 1-8; May 15, 2011; © 2011 Landes Bioscience RB1 and p53 at the crossroad of EMT and triple negative breast cancer Zhe Jiang,1 Robert Jones,1 Jeff C. Liu,1 Tao Deng,1 Tyler Robinson,1 Philip E.D. Chung,1 Sharon Wang,1 Jason I. Herschkowitz,2 Sean E. Egan,3 Charles M. Perou4 and Eldad Zacksenhaus1,* 1Division of Cell and Molecular Biology; Toronto General Research Institute; University Health Network; Toronto, Ontario, Canada; 2Department of Molecular and Cellular Biology; Baylor College of Medicine; Houston, TX USA; 3Program in Developmental and Stem Cell Biology; The Hospital for Sick Children; Department of Molecular Genetics; University of Toronto; Toronto, Ontario, Canada; 4Lineberger Comprehensive Cancer Center; Department of Genetics and Pathology; University of North Carolina at Chapel Hill; Chapel Hill, NC USA riple negative breast cancer (TNBC) NEU-positive and Triple Negative (TN) Tis a heterogeneous disease that tumors, the latter of which do not express includes Basal-like and Claudin-low hormone receptors or HER2.1-7 TNBC tumors. The Claudin-low tumors are affects 15–30% of patients. By IHC it can enriched for features associated with be further divided into Basal-like breast epithelial-to-mesenchymal transition cancer and non-basal tumors, some of (EMT) and possibly for tumor initiating which exhibit features of EMT.8 Basal-like cells. Primary TNBCs respond relatively BCs express the basal cytokeratins (CK) well to conventional chemotherapy; CK5/6, CK14, CK17, and/or epidermal © 2011 Landes Bioscience. Landes ©2011 however, metastatic disease is virtually growth factor receptor (EGFR), whereas incurable.
    [Show full text]
  • A Thyroid Hormone Receptor Coactivator Negatively Regulated by the Retinoblastoma Protein
    Proc. Natl. Acad. Sci. USA Vol. 94, pp. 9040–9045, August 1997 Biochemistry A thyroid hormone receptor coactivator negatively regulated by the retinoblastoma protein KAI-HSUAN CHANG*†,YUMAY CHEN*†,TUNG-TI CHEN*†,WEN-HAI CHOU*†,PHANG-LANG CHEN*, YEN-YING MA‡,TERESA L. YANG-FENG‡,XIAOHUA LENG§,MING-JER TSAI§,BERT W. O’MALLEY§, AND WEN-HWA LEE*¶ *Department of Molecular Medicine and Institute of Biotechnology, University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, TX 78245; ‡Department of Genetics, Obstetrics, and Gynecology, Yale University School of Medicine, New Haven, CT 06510; and §Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030 Contributed by Bert W. O’Malley, June 9, 1997 ABSTRACT The retinoblastoma protein (Rb) plays a E2F-1, a transcription factor important for the expression of critical role in cell proliferation, differentiation, and devel- several genes involved in cell cycle progression from G1 to S opment. To decipher the mechanism of Rb function at the (18). Rb inhibits E2F-1 activity by blocking its transactivation molecular level, we have systematically characterized a num- region (19–21). In contrast, Rb has been shown to have the ber of Rb-interacting proteins, among which is the clone C5 ability to increase the transactivating activity of the members described here, which encodes a protein of 1,978 amino acids of the CCAATyenhancer binding protein (CyEBP) family, and with an estimated molecular mass of 230 kDa. The corre- to be required for CyEBPs-dependent adipocyte and mono- sponding gene was assigned to chromosome 14q31, the same cytes differentiation (16–17).
    [Show full text]
  • Aberrant Cytoplasmic Accumulation of Retinoblastoma Protein in Basal Cells May Lead to Increased Survival in Malignant Canine Mammary Tumours
    Original Paper Veterinarni Medicina, 59, 2014 (2): 76–80 Aberrant cytoplasmic accumulation of retinoblastoma protein in basal cells may lead to increased survival in malignant canine mammary tumours S. Gautam, N.K. Sood, K. Gupta Guru Angad Dev Veterinary and Animal Sciences University Ludhiana, Ludhiana, Punjab, India ABSTRACT: The retinoblastoma susceptibility geneRB-1 is a tumour suppressor gene that encodes a protein (Rb) that regulates the transition from the G1 phase to the S phase of the cell cycle. Inactivation of the Rb gene has been shown in a variety of human tumours, including breast, ovarian, hepatic, prostatic, and endometrial carcinomas. Although Rb protein is normally expressed in the nuclei of healthy cells, during carcinogenesis there is a partial or complete loss of nuclear expression. Recently, some reports have indicated aberrant cytoplasmic expression of Rb protein. However, little is known about its cytoplasmic expression and significance as a prognostic marker in canine mammary tumours (CMT). The present study was performed on 36 malignant CMT cases in order to assess the mutational status and prognostic significance of Rb in primary malignant CMT. We report an almost complete loss of nuclear expression of Rb protein with corresponding gain of aberrant cytoplasmic expression in basal/myoepithelial cells in CMT. Strikingly, our analysis reveals a significant positive correlation between survival time and cytoplasmic expression of Rb protein in basal cells. Moreover, cytoplasmic expression of Rb protein in basal cells was also correlated with tumour grade and stage. Keywords: canine mammary tumour; dogs; immunohistochemistry; retinoblastoma protein List of abbreviations CMT = canine mammary tumour, RB = retinoblastoma gene, Rb = retinoblastoma protein Breast cancer is the most frequent malignant tu- suppressors.
    [Show full text]
  • Cortical Foxp2 Supports Behavioral Flexibility and Developmental Dopamine D1 Receptor Expression
    bioRxiv preprint doi: https://doi.org/10.1101/624973; this version posted May 2, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Cortical Foxp2 supports behavioral flexibility and developmental dopamine D1 receptor expression Marissa Co, Stephanie L. Hickey, Ashwinikumar Kulkarni, Matthew Harper, Genevieve Konopka* Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, USA *Correspondence: [email protected] Contact information Genevieve Konopka, Ph.D. Department of Neuroscience, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., ND4.300, Dallas, TX 75390-9111 TEL: 214-648-5135, FAX: 214-648-1801, Email: [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/624973; this version posted May 2, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Abstract FoxP2 encodes a forkhead box transcription factor required for the development of neural circuits underlying language, vocalization, and motor-skill learning. Human genetic studies have associated FOXP2 variation with neurodevelopmental disorders (NDDs), and within the cortex, it is coexpressed and interacts with other NDD-associated transcription factors. Cortical Foxp2 is required in mice for proper social interactions, but its role in other NDD-relevant behaviors and molecular pathways is unknown.
    [Show full text]
  • Androgens Repress Bcl-2 Expression Via Activation of the Retinoblastoma (RB) Protein in Prostate Cancer Cells
    Oncogene (2004) 23, 2161–2176 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $25.00 www.nature.com/onc Androgens repress Bcl-2 expression via activation of the retinoblastoma (RB) protein in prostate cancer cells Haojie Huang1, Ofelia L. Zegarra-Moro1, Douglas Benson1 and Donald J Tindall*,1 1Departments of Urology and Biochemistry/Molecular Biology, Mayo Clinic/Foundation, Rochester, MN 55905, USA The oncogene Bcl-2 is upregulated frequently in prostate function of the normal prostate gland, but also for the tumors following androgen ablation therapy,and Bcl-2 proliferation and survival of androgen-sensitive and - overexpression may contribute to the androgen-refractory refractory prostate cancer cells (Grossmann et al., 2001; relapse of the disease. However,the molecular mechanism Huang and Tindall, 2002; Zegarra-Moro et al., 2002). underlying androgenic regulation of Bcl-2 in prostate Surgical or pharmaceutical ablation of testicular andro- cancer cells is understood poorly. In this study,we gens has been the most effective treatment of metastatic demonstrated that no androgen response element (ARE) prostate cancer since 1941 (Huggins and Hodge, 1941). was identified in the androgen-regulated region of the P1 Both normal and malignant epithelial cells of the promoter of Bcl-2 gene,whereas,we provided evidence prostate undergo programmed cell death (apoptosis)in that the androgenic effect is mediated by E2F1 protein the absence of androgens (Isaacs, 1984; Kyprianou et al., through a putative E2F-binding site in the promoter. We 1990). However, a majority of prostate cancer patients further demonstrated that retinoblastoma (RB) protein usually relapse with tumors becoming refractory to plays a critical role in androgen regulation of Bcl-2.
    [Show full text]
  • Identification of a 60-Kilodalton Rb-Binding Protein, RBP60, That
    MOLECULARi AND CELLULAR BIOLOGY, OCt. 1992, p. 4327-4333 Vol. 12, No. 10 0270-7306/92/104327-07$02.00/0 Copyright © 1992, American Society for Microbiology Identification of a 60-Kilodalton Rb-Binding Protein, RBP60, That Allows the Rb-E2F Complex To Bind DNA SUBIR KUMAR RAY, MAY ARROYO, SRILATA BAGCHI, AND PRADIP RAYCHAUDHURI* Department ofBiochemistry (MIC 536), University ofIllinois at Chicago, Box 6998, Chicago, Illinois 60680 Received 11 March 1992/Returned for modification 17 April 1992/Accepted 1 July 1992 Several reports have indicated that the product of the retinoblastoma gene (Rb) complexes with the transcription factor E2F. We present evidence that the DNA-binding of the Rb-E2F complex involves another cellular factor. Addition of Rb to purified preparations of E2F does not generate an Rb-E2F complex that can bind DNA, and in fact, we see an inhibition of the DNA-binding ability of E2F. On the other hand, addition of Rb to cruder preparations of E2F results in the formation of an Rb-E2F complex (E2Fr) that can bind DNA and produces a distinct complex in gel retardation assays. We have identified and purified a 60-kDa protein that allows the Rb-E2F complex to bind DNA, and we show that this 60-kDa protein exerts its effect by directly interacting with Rb. The retinoblastoma gene is one of the best characterized contrast, Rb induces formation of a complex involving E2F tumor suppressor genes. The protein encoded by the reti- and its cognate sequence. By fractionating extracts from noblastoma gene, Rb, binds several DNA tumor virus onco- mouse L cells, we have identified and purified a factor, proteins (12, 15, 16) including adenovirus ElA, simian virus RBP60, that protects E2F from Rb inhibition and allows 40 T antigen, and papillomavirus E7 protein.
    [Show full text]